A

pc(icgd1wu©

K

1

nilrrdpdnd

0d

Talanta 71 (2007) 391–396

Development of a long-life capillary enzyme bioreactorfor the determination of blood glucose

Ja-an Annie Ho a,b,∗, Li-chen Wu b, Nien-Chu Fan a, Ming-Shih Lee c,d,Hung-Yi Kuo b, Chung-Shi Yang b

a Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwanb Department of Applied Chemistry, National Chi-Nan University, Puli, Nantou 545, Taiwan

c Department of Medical Laboratory, Taichung Veterans General Hospital, Taichung 407, Taiwand Department of Medical Technology, Chung Shan Medical University, Taichung 402, Taiwan

Received 11 January 2006; received in revised form 11 April 2006; accepted 11 April 2006Available online 5 June 2006

bstract

A long-life capillary enzyme bioreactor was developed that determines glucose concentrations with high sensitivity and better stability thanrevious systems. The bioreactor was constructed by immobilizing glucose oxidase (GOx) onto the inner surface of a 0.53 mm i.d. fused-silicaapillary that was part of a continuous-flow system. In the presence of oxygen, GOx converts glucose to gluconic acid and hydrogen peroxideH2O2). Hydrogen peroxide detection was accomplished using an amperometric electrochemical detector. The integration of this capillary reactornto a flow-injection (FIA) system offered a larger surface-to-volume ratio, reduced band-broadening effects, and reduced reagent consumptionompared to packed column in FIA or other settings. To obtain operational (at ambient temp) and storage (at 4 ◦C) stability for 20 weeks, thelucose biosensing system was prepared using an optimal GOx concentration (200 mg/mL). This exhibited an FIA peak response of 7 min and a

etection limit of 10 �M (S/N = 3) with excellent reproducibility (coefficient of variation, CV < 0.75%). It also had a linear working range from01 to 104 �M. The enzyme activity in this proposed capillary enzyme reactor was well maintained for 20 weeks. Furthermore, 20 serum samplesere analyzed using this system, and these correlated favorably (correlation coefficient, r2 = 0.935) with results for the same samples obtainedsing a routine clinical method. The resulting biosensing system exhibited characteristics that make it suitable for in vivo application.

2006 Elsevier B.V. All rights reserved.

ood g

ibtts[hsmt

eywords: Continuous-flow system; Flow-injection analysis; FIA; Glucose; Bl

. Introduction

Human body needs to maintain blood glucose within a veryarrow range of 70–110 mg/dL. People who have diabetes orncreased fasting levels of glucose have elevated blood glucoseevels because of an inability to use insulin properly. This is ofteneferred to as insulin resistance. Statistics show that diabetes haseached epidemic levels in the U.S. because of increased inci-ence among older Americans, as well as more obesity in theopulation. About 2200 people are diagnosed with diabetes each

ay, but about one-third of the individuals who have diabetes areot aware of it until one of its life-threatening complications haseveloped. Diabetes results in long-term health consequences,

∗ Corresponding author. Tel.: +886 3571 5131x31286; fax: +886 3 571 1082.E-mail address: jaho@mx.nthu.edu.tw (J.-a.A. Ho).

[tdelc

039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2006.04.023

lucose; Glucose oxidase

ncluding cardiovascular disease, nephropathy, neuropathy, dia-etic retinopathy and blindness. Recent research has indicatedhat hyperglycemia is common in critically ill patients, even inhose without diabetes mellitus. It has been reported that aggres-ive glycemic control may reduce mortality in this population1]. However, the relationship among mortality, the control ofyperglycemia, and the administration of exogenous insulin istill unclear. Therefore, it is very important to have a simpler,ore-stable, and more-sensitive method that allows the moni-

oring of blood glucose in clinics and laboratories.The glucose sensor reported by Clark and Lyons in 1962

2] has generally been recognized as the first biosensor. Sincehen, many types of sensors have been developed for medical

iagnosis applications. The use of glucose oxidase (GOx)-basedlectrodes is a well-established method of detection for in vivoevels of circulating glucose [3–5]. In this approach, glucose isonverted to gluconic acid and easily detectable hydrogen perox-

3 lanta

iouitsWictepohtspwiMeiddoac

nlefitec

aatittpacfmag

2

2

r3

sea(oiwTo(Hf

2

2

tvlcdsoetfim

2

92 J.-a.A. Ho et al. / Ta

de (H2O2) by the enzyme glucose oxidase. The process requiresxygen as a cosubstrate. The produced H2O2 is then measuredsing a charged platinum electrode surface. Hydrogen perox-de has become by far the most widely used method of signalransduction in enzyme biosensors, and a majority of all biosen-ors (65%) use hydrogen peroxide detection [6]. According to

ilson and Thevenot [7], the construction of a hydrogen perox-de sensor usually involves a platinum anode and a silver/silverhloride cathode. When the anode is poised at +0.6–0.7 V [8],he plateau of oxidation of peroxide is reached at the anode. Thenzyme employed in the construction of hydrogen peroxide-roducing biosensors frequently involves the immobilizationf oxido-reductases to the surface of the sensor by glutaralde-yde cross-linking [8]. Other methods have been reported forhe immobilization of enzyme, such as physical deposition ontoolid supports, covalent binding [9], and entrapment within aolyer matrix [10]. In recent years, sol–gel technology has beenidely used to entrap enzyme for different uses [11–14], because

t retains better enzyme activity compared to the free enzyme.atrixes are usually prepared under ambient conditions and

xhibit tunable porosity, high thermal stability, and chemicalnertness [12]. However, the silica sol–gel matrixes have somerawbacks, including fragility, complicated preparation proce-ures, and a tendency to be hydrolyzed at high acidity, whichften results in the loss of enzyme stability and also limits theirpplication and feasibility in the development of electrochemi-al sensors [15,16].

Many methods have been developed in an effort to find aoninvasive detection system for circulating glucose at in vivoevels, including ultrasound-assisted transdermal monitoring,lectromagnetic-based sensor, and fluorescence-affinity hollow-ber sensors [17–27]. Other methods of glucose determination

hat have been reported include those based on a geneticallyngineered protein [28], on concanavalin A [29], and on a micro-antilever [30].

In this work we develop a longer-life capillary enzyme biore-ctor for the determination of glucose. The greatly improvedctivity and stability of this new enzyme bioreactor is facili-ated by the direct attachment of GOx to the wall of a 530 �m.d. fused-silica capillary. To the best of our knowledge this ishe first demonstration of the capillary glucose oxidase bioreac-or with improved enzyme stability and longer shelf life, whichrovides a higher surface-to-volume ratio, maximizing the inter-ction between glucose and GOx compared to bead-packedolumn. The results from blood sample analysis promised wellor the use of this biosensing system in online blood glucoseonitoring of critically ill patients before and after surgical oper-

tions. Reduced mortality can therefore be achieved by intensivelycemic control.

. Materials and methods

.1. Reagents and materials

d-(+)-Glucose, glucose oxidase (glucose: oxygen oxido-eductase E.C. 1.1.3.4, from Aspergillus niger, 181.6 U/mg),-glycidoxy propyltrimethoxysilane, potassium carbonate,

sp

71 (2007) 391–396

odium metaperiodate, sodium cyanborohydride and tri-thanolamine, glycine, and Trizma® Base tris[hydroxymethyl]minomethane (Tris) were obtained from Sigma Chemicals Co.St. Louis, MO). The fused-silica capillary (0.53 mm i.d.) wasbtained from Alltech (Deerfield, IL). All other inorganic chem-cals and organic solvents were of reagent grade or better andere purchased from Aldrich Chemical Co. (St. Louis, MO).he pre-analyzed blood plasma samples from patients werebtained fresh from the Veteran General Hospital—TaichungVGHTC). The use of these samples in no way contradicts theelsinki Declaration. De-ionized distilled water was obtained

rom a Milli-Q system (Milford, MA).

.2. Methods

.2.1. Capillary modificationFor a sensitive flow-injection analysis (FIA) enzyme reac-

or, we required a high-enzyme loading comparable to the deadolume of the bed. For such an enzyme assay, the immobi-ization support must be rigid and have a mild, very stable,ovalent immobilization chemistry. Our group has previouslyemonstrated the successful immobilization of biomoleculesuch as antibody without loss of activity and decreased stabilityn the inner surface of capillary column precoated with a glyc-rylpropyl layer to minimize the adsorption of the analyte. Inhe current study the microcapillary enzyme reactor was modi-ed based on previously described procedures [31–36]. Detailedodification procedures were as follows:

Step 1: The 85 cm fused-silica capillary (0.53 mm i.d.) wastreated with 1 M NaOH overnight.Step 2: 1 M HCl and distilled water were used torinse the capillary, which was subsequently filled with 3-glycidoxypropyltrimethoxysilane (GPTMS) and heated at90 ◦C for 2 h.Step 3: The capillary was rinsed and treated with 10 mM sul-furic acid at 90 ◦C for 10 min to convert the residual epoxygroups to diols.Step 4: After washing with distilled water, diols were cleavedand oxidized to aldehydes with sodium metaperiodate contain-ing potassium carbonate at room temperature for 2 h.Step 5: 190 �L of GOx (200 mg/mL) and sodium cyanboro-hydride (5 mg/mL) in 0.1 M phosphate buffer (pH 7.3) werepassed slowly into the capillary and incubated overnight toreduce the Schiff base.Step 6: The capillary was rinsed with 0.2 M triethanolaminebuffer (pH 8.2), 1 M NaCl, 0.1 M glycine/HCl buffer (pH 2.5),and Tris buffered saline (TBS), pH 7.0, sequentially. Finally,the capillary enzyme reactor, filled with TBS (pH 7.0), wasthen stored at 4 ◦C until use. In this way glucose oxidase wascovalently attached on the inner wall of capillary column.

.3. Flow-injection analysis system

The flow-injection analysis system (schematic diagramhown at Fig. 1) consists of a Hewlett Packard 1050 HPLCump (Agilent, Foster City, CA) at the inlet of the capillary glu-

J.-a.A. Ho et al. / Talanta 71 (2007) 391–396 393

F ood g

calC(fu0Cc

2CwaIeefiM

2

wioriwati0tmmo

aHoauTod

2

pstwa

3

3b

cae(

tsmvhl

ig. 1. A schematic diagram of enzymatic flow injection analytical system for bl

ose biosensing system that maintains a flow rate of 0.3 mL/minnd a Rheodyne (Model 7725) injector with a 20 �L sampleoop (Rainin, Emeryville, CA) for injection of the samples.ommercially available polyetheretherketone (PEEK) tubing

0.020 in. i.d.) and standard fingertight fittings were purchasedrom Upchurch Scientific Inc. (Oak Harbor, WA). The carriersed was 0.1 M potassium phosphate buffer (pH 7.2) containing.1 M NaCl that was vacuum-filtered before use. A HW-2000hromatography workstation, used for data collection, was pur-hased from Great Tide Instrument Co. (Taipei, Taiwan).

Final signal integration was performed using the HW-000 Chromatography workstation system running on an Inteleleron 2.20 GHz computer. All electrochemical measurementsere performed on an electrochemical analyzer (model CL-4C

mperometric detector) obtained from BAS (West Lafayette,N). A BAS model CC-5E electrochemical flow cell wasmployed in these measurements. The conventional three-lectrode system was made up of a dual platinum electrodeor thin-layer cross-flow cell (model MF-1012, BAS) as work-ng electrode (3 mm in diameter), Ag|AgCl as reference (model

W-2078, BAS), and steel wire as counter electrode.

.4. Experimental procedures

For the determination of glucose, 20 �L of glucose standardsere injected into the carrier stream and subjected to flow-

njection analysis using the constructed microcapillary glucosexidase enzyme reactor as described above, where the catalyticeaction involves glucose oxidation to produce hydrogen perox-de. The amperometric signal produced by hydrogen peroxideas measured by the amperometric detector in the system, which

pplied a potential to the electrochemical cell and monitoredhe resulting electrochemical reaction. The dual platinum work-ng electrode was initially prepared by polishing for 3 min with.05 �m diamond polish on a polishing disk. After polishing,

he dual electrode surface was rinsed with distilled water and

aintained at 0.350 V versus Ag|AgCl for hydrogen peroxideeasurements. The electrochemical oxidation of hydrogen per-

xide at the dual 3 mm platinum electrode was measured with an

soia

lucose. WE: working electrode; RE: reference electrode; AE: counter electrode.

mperometric detector, and the current output was also stored onW-2000. The height of a given FIA peak reflects the numberf moles injected onto the capillary enzyme reactor. At a givennalyte concentration, the peak height varies with sample vol-me, which is determined by the volume of the injection loop.he calibration curves for each assay were expressed in termsf the injected molar content in order to determine the linearynamic range of the capillary FIA system for glucose.

.5. Real-sample analysis

Fresh human serum (20 �L) was diluted with 180 �L of 0.1 Motassium phosphate buffer (pH 7.2) containing 0.1 M NaCl andubsequently injected via the valve. The results obtained usinghis capillary-based method were compared to those obtainedith a clinically used glucose analyzer (Hitachi 7170 automated

nalyzer).

. Results and discussion

.1. Optimization of parameters and characterization of theiosensing system

A series of experiments was performed to establish theonditions for maximum peak height. The applied volt-ge (0.300–0.500 V), sample injection volume (5–20 �L),nzyme concentration (100–200 mg/mL), and flow rate0.1–0.5 mL/min) were investigated.

To evaluate the effect of the voltage on the sensitivity ofhe biosensing system, different voltages were applied to theystem. The voltages varied from 0.300 to 0.500 V. The maxi-um sensitivity was achieved at an applied voltage of 0.500 V

ersus Ag|AgCl using model glucose standards. However, thisigh voltage suffered from a low signal-to-noise ratio prob-em, and there was severe interference from the medium during

erum sample analysis (Fig. 2). Oxidation of hydrogen per-xide at higher voltage (at a platinum electrode) is prone tonterference from many other electroactive substances, such asscorbic acid and uric acid; however, oxidation signals obtained

394 J.-a.A. Ho et al. / Talanta 71 (2007) 391–396

afmHwdfbTATwittTpcwwtborAw

3

t

G

Isampwpn

Fs

t

H

Tacl1oslispsevseTewFfto fouling by protein components in physiological fluids, noserious interference was found when our model serum solution(BSA spiked in glucose standard solution at final concentrationof 7 mg/mL) was tested.

Fig. 2. The effect of applied voltage on real-sample analysis.

t lower potential minimize these interference effects. There-ore, a voltage of 0.350 V was selected, and the system becameore tolerant of interference. Although results obtained fromitachi 7170 autoanalyzer and proposed FIA system correlatedell, differences were still observed between these two sets ofata. The accuracy of the proposed system should be satisfactoryor screening purpose. The injected-sample volume was variedy changing sample loop size relative to the injection valve.he peak height increased with increasing injected-sample size.sample volume of 20 �L was selected as a suitable volume.

he enzyme concentration for the immobilization on the innerall of capillary was varied from 100 to 200 mg/mL. The max-

mum peak height was obtained with GOx at 200 mg/mL. Dueo the relatively high cost of glucose oxidase, enzyme concen-ration higher than 200 mg/mL was not considered in this study.he length of bioreactor (25–100 cm) was also an investigatedarameters. The longer the bioreactor, the higher signal outputould be obtained. However, broaden peaks were often foundhen length of bioreactor was longer than 85 cm. The flow rateas a very important parameter of the proposed system because

he slower flow allowed sample glucose to react with immo-ilized enzyme more completely, and therefore higher signalutput could be collected; however, the slow flow rate oftenesulted in peak broadening and limited sample throughput.fter considering all of these factors, a flow rate of 0.3 mL/minas chosen for acceptable peak height and sample throughput.

.2. Assay performance

The present enzyme-based capillary glucose biosensing sys-em was based on glucose oxidase.

lucose + O2 ↔ H2O2 + gluconicacid

n the reaction sequence shown above, glucose oxidase cataly-es the oxidation of �-d-glucose to gluconic acid, using oxygens the electron acceptor. Since the gluconic acid level cannot beeasured by the change in pH [8], the oxidation of hydrogen

eroxide was measured by means of a charged platinum-basedorking electrode surface [3]. Based on earlier research [37–40],otassium phosphate buffered saline (pH 7.2) with a similarature to physiological fluids was selected as the medium solu-

FG

ig. 3. Reproducibility of the signals generated by five replicates of a glucosetandard (concentration = 10 mM).

ion for the glucose standards.

2O2+0.350−0.650V vs Ag/AgCl−→ O2 + 2H+ + 2e−

he amperometric signal was obtained upon the injection of vari-ble glucose concentrations into the electrode cell under flowonditions, no signal was found in the absence of glucose. Theinear response was observed between 10−2 mM (10 �M) and0 mM of glucose standard solution with the regression equationf amperometric signal = 49.879 mM + 2.21 (R2 = 1.000). Theensing system was used for an average of 8 h a day, and the capil-ary enzyme reactor remained stable for at least 120 days at 25 ◦Cn operation condition and for the remainder of the time at 4 ◦C intorage conditions. The repeatability and reproducibility of theroposed system was examined by injecting a 10 mM glucosetandard. The uniformity of the FIA-amperometric peaks gen-rated by five replicate injections is shown in Fig. 3. The largestalue for the coefficient of variation (CV) for five replicate mea-urements was 0.75%, indicating that the reproducibility of thisnzyme-based capillary glucose sensing system is acceptable.he proposed method was also validated for its accuracy bymploying interday studies for 5 days where a glucose standardas measured, also at a concentration of 10 mM. As shown inig. 4, the coefficient of variation based on the peak height wasound to be 3.05%. Additionally, though Pt electrode is prone

ig. 4. Interday deviation for the enzyme-based capillary biosensing system.lucose standard concentration: 10 mM.

J.-a.A. Ho et al. / Talanta

Fig. 5. Linear regression of the correlation between glucose results measuredby the proposed capillary glucose biosensing system and reference clinical mea-smE

3

w(mcpw(TibccorcHtpaa

4

olObsittaw

fldi

sto1afcbdiobsdcttaasoc

A

iTs

R

[

[[[

urements of blood samples obtained from 20 patients. X-axis: results of clinicaleasurements; Y-axis: results from the capillary glucose biosensing system.ach point represents the mean of three measurements.

.3. Application

The comparison study in measuring blood glucose of patientsith proposed system and clinically used glucose analyzer

Hitachi 7170) is very important in validating a new analyticalethodology towards its clinical applications. In this study, glu-

ose was determined in human serum from adults by using theroposed system. Prior to analysis the sample serum was dilutedith 0.1 M potassium phosphate buffer containing 0.1 M NaCl

pH 7.2) and centrifuged at 2000 × g at room temp for 5 min.wenty microliters was taken directly from the supernatant and

njected into the system. Linear regression of the correlationetween blood glucose results measured by the enzyme-basedapillary glucose biosensing system and a clinically used glu-ose analyzer (Hitachi 7170) is shown in Fig. 5. The largest valuef the CV for five replicate injections was 6.05%. The best-fitegression line of the average of enzyme-based capillary glu-ose sensing system versus that determined using the clinicalitachi 7170 automated analyzer indicated a strong correla-

ion between the two data sets (r2 = 0.935). This shows that theroposed system is comparable to the Hitachi 7170 automatednalyzer presently used in the Medical Laboratory Departmentt Taichung Veterans General Hospital.

. Conclusions

In summary, we have demonstrated the successful attachmentf glucose oxidase to the inner wall of a fused-silica capil-ary while retaining enzymatic activity for more than 120 days.ur study has shown that an enzyme-based capillary glucoseiosensing system was developed based on flow-injection analy-is with amperometric detection. Operational and storage stabil-ty for greater than 4 months permitted the measurement of more

han 300 samples. The glucose biosensing system prepared usinghe optimal GOx concentration (200 mg/mL) exhibited a FIA-mperometric current response at 7 min. The sample throughputas about 9/h, and the reagent consumption was reduced. This

[[[

71 (2007) 391–396 395

ow-injection type of sensing system holds promise for theetermination of glucose content in clinical samples as well asn fruit juice.

The proposed glucose sensing system was found to be respon-ive to glucose over a wide range of concentrations and hashe following characteristics: a detection limit of 10 �M (basedn the signal-to-noise characteristics, S/N = 3), linearity up to04 �M, and reproducibility of under 0.75% coefficient of vari-tion. These results clearly show the usefulness of this platformor direct detection of glucose and clinical diagnosis withoutomplicated sample preparation and labeling. This proposediosensing system has demonstrated its feasibility as a means ofetermining blood glucose in serum. The integration of this cap-llary system into a flow-injection system offers the advantagesf a large surface-to-volume ratio, laminar flow, and reducedand-broadening effects compared to previous packed-columnystems. These all help to increase the sensitivity and repro-ucibility of this capillary glucose measurement system. Theurrent focus of our group is to use an enzyme-immobilizationechnique on microfluidic enzyme chips to improve the samplehroughput. Future efforts will include attempts to incorporaten insulin biosensor into the present sensing system that willllow the parallel measurement of glucose and insulin. This willimplify studies on whether blood glucose level or the amountf insulin administered is associated with reduced mortality inritically ill patients.

cknowledgements

This work was supported by the National Science Counciln Taiwan, ROC, under grant NSC 91-2113-M-260-011 and theaichung Veterans General Hospital-National Chi-Nan Univer-ity Joint Research Program under grant VGHCN 92-72-02.

eferences

[1] S.J. Finney, C. Zekveld, A. Elia, T.W. Evans, JAMA 290 (2003)2041.

[2] L.C. Clark Jr., C. Lyons, Ann. N Y Acad. Sci. 102 (1962) 29.[3] G.G. Guilbault, G.J. Lubrano, Anal. Chim. Acta 64 (1973) 439.[4] Y. Zhang, G.S. Wilson, Anal. Chim. Acta 281 (1993) 513.[5] Y. Hu, G.S. Wilson, J. Neurochem. 68 (1997) 1745.[6] J. Davis, D.H. Vaughan, M.F. Cardosi, Enzyme Microbiol. Technol. 17

(1995) 1030.[7] G.S. Wilson, D.R. Thevenot, in: A.E.G. Cass (Ed.), Biosensor, A Practical

Approach, IRL Press, Oxford, UK, 1990, pp. 1–17.[8] J. Woodward, in: A. Mulchandani, K.R. Rogers (Eds.), Enzyme and Micro-

bial Biosensor, Techniques and Protocols, Humana Press, New Jersey,USA, 1998, pp. 67–79.

[9] K.T. Lee, C.C. Akoh, Food Rev. Int. 14 (1998) 17.10] D. Parra-Diaz, D.P. Brower, M.B. Medina, G.J. Piazza, Biotechnol. Appl.

Biochem. 18 (1993) 359.11] A. Hsu, T.A. Foglia, S. Shen, Biotechnol. Appl. Biochem. 31 (2000) 179.12] J. Yu, H. Ju, Anal. Chem. 74 (2002) 3579.13] Th. Noguer, D. Szydlowska, J.-L. Marty, M. Trojanowicz, Polish J. Chem.

78 (2004) 1679.14] Y. Tang, E.C. Tehan, Z. Tao, F.V. Bright, Anal. Chem. 75 (2003) 2407.15] Q. Chen, G.L. Kenausis, A. Heller, J. Am. Chem. Soc. 120 (1998) 4582.16] O. Lev, M. Narvaez, E. Dominguez, I. Kataki, J. Electroanal. Chem. 425

(1997) 1.

3 lanta

[

[

[[

[[

[[[[

[

[

[[[[[[[

96 J.-a.A. Ho et al. / Ta

17] M. Gourzi, A. Rouane, R. Guelaz, M. Nadi, F. Jaspard, J. Med. Eng. Tech-nol. 27 (2003) 276.

18] J. Kost, S. Mitragotri, R. Gabbay, M. Pishko, R. Langer, Nature 6 (2000)347.

19] R. Ballerstadt, J.S. Schultz, Anal. Chem. 72 (2000) 4185.20] M. Gerritsen, J.A. Jansen, A. Kros, R.J.M. Nolte, J.A. Lutterman, Invest.

Surg. 11 (1998) 163.21] R. Ballerstadt, J.S. Schultz, Anal. Chim. Acta 345 (1997) 203.22] E. Wilkins, P. Atanasov, B.A. Muggenburg, Biosens. Bioelectron. 10 (1995)

485.23] D.L. Meadows, J.S. Schultz, Anal. Chim. Acta 280 (1993) 21.

24] S. Mansouri, J.S. Schultz, Biotechnology 2 (1984) 885.25] P. Abel, A. Muller, U. Fischer, Biomed. Biochim. Acta 3 (1984) 577.26] J.C. Pickup, D.R. Thevenot, European achievements in sensor research

dedicated to in vivo monitoring, in Advances in biosensors, supplement 1,JAI Press, London, UK, 1993, pp. 273–288.

[[[[[

71 (2007) 391–396

27] S. Jung, J.R. Trimarchi, R.H. Sanger, P.J.S. Smith, Anal. Chem. 73 (2001)3759.

28] L. Tolosa, I. Gryczynski, L.R. Eichhorn, J.D. Dattelbaum, F.N. Castellano,G. Rao, J.R. Lakowicz, Anal. Biochem. 267 (1999) 114.

29] R.J. Russell, M.V. Pishko, Anal. Chem. 71 (1999) 3126.30] J. Pei, F. Tian, T.B. Thundat, Anal. Chem. 76 (2004) 292.31] P. Larsson, Methods Enzymol. 104 (1984) 212.32] M. de Frutos, S.K. Paliwal, F.E. Regnier, Anal. Chem. 65 (1993) 2159.33] M.Y. Lee, R.A. Durst, J. Agric. Food Chem. 44 (1996) 4032.34] J.A. Ho, R.A. Durst, Anal. Chim. Acta 414 (2000) 61.35] F.E. Regnier, R. Noel, J. Chromatogr. Sci. 14 (1976) 316.

36] L.C. Wu, C.M. Cheng, Anal. Biochem. 346 (2005) 234.37] J. Wang, M. Chatrathl, A. Ibanez, Analyst 126 (2001) 1203.38] M.G. Booutelle, L.K. Fellows, C. Cook, Anal. Chem. 64 (1992) 1790.39] T. Hoshi, J. Anzai, T. Osa, Anal. Chem. 67 (1995) 770.40] S. Hrapovic, J.H.T. Luong, Anal. Chem. 75 (2003) 3308.